Sphingomyelin synthase–related protein SMSr is a phosphatidylethanolamine phospholipase C that promotes nonalcoholic fatty liver disease

Sphingomyelin synthase (SMS)–related protein (SMSr) is a phosphatidylethanolamine phospholipase C (PE-PLC) that is conserved and ubiquitous in mammals. However, its biological function is still not clear. We previously observed that SMS1 deficiency–mediated glucosylceramide accumulation caused nonalcoholic fatty liver diseases (NAFLD), including nonalcoholic steatohepatitis (NASH) and liver fibrosis. Here, first, we evaluated high-fat diet/fructose-induced NAFLD in Smsr KO and WT mice. Second, we evaluated whether SMSr deficiency can reverse SMS1 deficiency–mediated NAFLD, using Sms1/Sms2 double and Sms1/Sms2/Smsr triple KO mice. We found that SMSr/PE-PLC deficiency attenuated high-fat diet/fructose–induced fatty liver and NASH, and attenuated glucosylceramide accumulation–induced NASH, fibrosis, and tumor formation. Further, we found that SMSr/PE-PLC deficiency reduced the expression of many inflammatory cytokines and fibrosis-related factors, and PE supplementation in vitro or in vivo mimicked the condition of SMSr/PE-PLC deficiency. Furthermore, we demonstrated that SMSr/PE-PLC deficiency or PE supplementation effectively prevented membrane-bound β-catenin transfer to the nucleus, thereby preventing tumor-related gene expression. Finally, we observed that patients with NASH had higher SMSr protein levels in the liver, lower plasma PE levels, and lower plasma PE/phosphatidylcholine ratios, and that human plasma PE levels are negatively associated with tumor necrosis factor-α and transforming growth factor β1 levels. In conclusion, SMSr/PE-PLC deficiency causes PE accumulation, which can attenuate fatty liver, NASH, and fibrosis. These results suggest that SMSr/PE-PLC inhibition therapy may mitigate NAFLD.

Sphingomyelin synthase (SMS)-related protein (SMSr) is a phosphatidylethanolamine phospholipase C (PE-PLC) that is conserved and ubiquitous in mammals.However, its biological function is still not clear.We previously observed that SMS1 deficiency-mediated glucosylceramide accumulation caused nonalcoholic fatty liver diseases (NAFLD), including nonalcoholic steatohepatitis (NASH) and liver fibrosis.Here, first, we evaluated high-fat diet/fructose-induced NAFLD in Smsr KO and WT mice.Second, we evaluated whether SMSr deficiency can reverse SMS1 deficiency-mediated NAFLD, using Sms1/ Sms2 double and Sms1/Sms2/Smsr triple KO mice.We found that SMSr/PE-PLC deficiency attenuated high-fat diet/fructose-induced fatty liver and NASH, and attenuated glucosylceramide accumulation-induced NASH, fibrosis, and tumor formation.Further, we found that SMSr/PE-PLC deficiency reduced the expression of many inflammatory cytokines and fibrosis-related factors, and PE supplementation in vitro or in vivo mimicked the condition of SMSr/PE-PLC deficiency.Furthermore, we demonstrated that SMSr/PE-PLC deficiency or PE supplementation effectively prevented membrane-bound β-catenin transfer to the nucleus, thereby preventing tumorrelated gene expression.Finally, we observed that patients with NASH had higher SMSr protein levels in the liver, lower plasma PE levels, and lower plasma PE/phosphatidylcholine ratios, and that human plasma PE levels are negatively associated with tumor necrosis factor-α and transforming growth factor β1 levels.In conclusion, SMSr/PE-PLC deficiency causes PE accumulation, which can attenuate fatty liver, NASH, and fibrosis.These results suggest that SMSr/PE-PLC inhibition therapy may mitigate NAFLD.
Mammalian phospholipase C (PLC) is a group of enzymes that cleave phospholipids to produce a diacylglycerol (DAG) and a phosphorylated molecule, such as phosphorylcholine and phosphorylethanolamine (7).We and others found that SMSr has PLC activity (6,8) and specifically hydrolyzes PE, making it a phosphatidylethanolamine PLC (PE-PLC) (6).We also found that SMS1 and SMS2 are two phosphatidylcholine (PC)-PLCs (9).Given the fact that (10-13) PC-PLC and phosphatidylinositol (PI)-PLCs (14) maintain PC and PI levels and regulate many important biological functions, SMSr-mediated PE-PLC activity should have an important biological function.
PE is the second most abundant phospholipid in mammalian tissues (15,16).PE plays an important part in autophagy, cell division, protein folding, and endoplasmic reticulum stress (17)(18)(19)(20).Abnormally high or low PE/PC ratios affect energy metabolism in various tissues and are linked to metabolic diseases, including nonalcoholic fatty liver diseases (NAFLD), insulin resistance, and atherosclerosis (21)(22)(23)(24)(25)(26).Despite its importance, the regulation of PE levels and the mechanisms linking PE changes to metabolic diseases are poorly understood.
A hallmark of NAFLD is triglyceride accumulation in the cytoplasm of hepatocytes, which arises from an imbalance between lipid acquisition and removal (27).The ratio (or index) of triglyceride and glucose in the circulation is a useful marker for screening simple fatty liver and nonalcoholic steatohepatitis (NASH) in humans (28,29).However, other lipids, including sphingolipids, also have important roles in the development of fatty liver and NASH (30,31).Ceramides and glycosphingolipids could be two groups of these factors (32,33).We recently reported that SMS1 deficiency leads to glucosylceramide (GluCer) accumulation, and this is one of the triggers promoting the development of fatty liver into NASH, and subsequently fibrosis and tumorigenesis (34).Since SMSr is a member of the SMS family, we would like to evaluate the effect of SMSr/PE-PLC on GluCer-induced NAFLD.
In this study, we showed that SMSr/PE-PLC deficiency attenuated dietary-induced or GluCer accumulation-induced NAFLD.

Effect of SMSr deficiency on high-fat diet/fructose-induced fatty liver and NASH
We confirmed SMSr is a PE-PLC: administration of adenovirus-SMSr significantly increased PE-PLC activity and decreased PE levels in the liver of WT mice, whereas Smsr-KO mice had significantly lower PE-PLC activity and higher PE levels in livers compared with controls (Figs. 1, A-C and S1, A and B).To induce fatty liver and NASH, we fed Smsr-KO and WT mice with a high-fat diet plus fructose in drinking water for 16 weeks.Starting from week one, Smsr-KO mice gained significantly less body weight than WT mice (Fig. 1D).SMSr deficiency significantly reduced the levels of plasma triglyceride and cholesterol (Fig. 1E).The Smsr-KO mice had significantly lower levels of liver triglyceride than WT mice (Fig. 1F), which was confirmed by Oil Red O staining of the liver sections (Fig. 1G).We also stained the sections with trichrome blue staining.Although we did not observe fibrosis in both sections, we found that SMSr deficiency also reduced lipid droplet size in the liver (Fig. 1H).Two enzyme markers for liver damage, alanine transaminase (ALT) and aspartate transaminase (AST), had significantly lower plasma activity levels in Smsr-KO mice than in WT mice (Fig. S2).
Next, we isolated liver from Smsr KO and WT mice and performed total RNA-seq.The volcano plot shows that the absence of SMSr in the liver altered the expression of 481 genes (padj < 0.05 and|FC|>1.5)including 236 upregulated and 245 downregulated genes (Fig 2A).Notably, PANTHER GO-slim analysis revealed that lipid metabolism-related biological processes were altered in Smsr KO mice (Fig. 2B), including fatty acid metabolism, lipid transport, and lipid metabolism.An overview of the top 35 upregulated and downregulated genes is given in Figure 2C.These changes could have direct or indirect effects on fatty liver and NASH.
We performed real-time quantitative PCR in liver samples to examine gene expression levels and investigate potential biological mechanisms.The results showed that Fsp27, PPARγ2, fatty acid synthase (FAS), Tnfα, and Timp1 expression levels were significantly reduced in Smsr-KO mice than those in WT mice (Fig. 2D).No other statistically significant changes in gene expression levels under SMSr/PE-PLC deficiency were detected (Fig. S3).Next, we examined protein levels of Fsp27, FAS, and PPARγ2 in the mouse liver.The results indicated that levels of Fsp27 protein but not FAS or PPARγ2 were significantly reduced (Fig. 2E).
We investigated the consequences of fatty liver by performing cytokine array analyses in mouse plasma and found that SMSr/PE-PLC deficiency-induced reductions of about 30 inflammatory cytokines (Fig. 2F).We performed ELISA to specifically measure the levels of plasma tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6), two well-known inflammatory cytokines.The results showed that both were significantly reduced (Fig. 2H).These combined results indicate that SMSr/PE-PLC deficiency attenuates high diet/fructose-induced fatty liver and NASH which could be associated with fibrosis or without fibrosis (35).However, we did not observe fibrosis in both WT and SMSr/PE-PLC KO mice.

Effect of SMSr deficiency on glucosylceramide-induced nonalcoholic steatohepatitis, liver fibrosis, and liver tumor
Our previous study reported that 3-month-old mice with Sms1 and Sms2 double KO (dKO) fed with a high-fat diet for 6 weeks had significantly higher lipid accumulation in liver than that of Sms2-KO or WT mice (34).Here, we prepared Effect of SMSr, as a PE-PLC, deficiency on NAFLD Sms1/Sms2/Smsr triple KO (tKO) mice and then fed WT, Sms1/Sms2-dKO, and Sms1/Sms2/Smsr-tKO mice (3-monthold, male and female) with high-fat diet for 6 weeks.The results showed that SMSr deficiency did not prevent lipid accumulation (Fig. S4, A and B).
Next, we utilized the dKO and the tKO mice on chow diet to perform the rest of the experiments.We measured different lipid levels in the liver of the dKO and the tKO mice and confirmed that there were no differences in sphingomyelin and ceramide (9) (Fig. S5).We also measured different subspecies of PE in the liver of WT, dKO, and tKO mice, and found that total and almost all tested individual PEs were significantly higher in tKO mice than in WT or dKO mice (Fig. 3, A and B).We confirmed that the dKO mice had significantly higher GluCer levels in the liver than the WT mice (34) (Fig. 3, C and D).Importantly, the tKO mice had significantly lower GluCer levels in the liver than the dKO mice (Fig. 3, C and D), which could be due to SMSr deficiency-mediated PE accumulation.We then treated the dKO mice with PE and found that PE supplementation significantly reduced glucosylceramide synthase (GCS) activity (Fig. 3E).These combined results suggest that SMSr/PE-PLC deficiency-mediated PE induction could reduce GCS activity thereby reducing GluCer levels in the liver.
Our previous study observed liver abnormalities in a 6month-old dKO mice that were on a chow diet compared with WT and Sms2-KO mice (34).The current study utilized mice that were ≥6-months old.We visualized hepatocyte basal membranes in WT, the dKO, and the tKO mice by immunostaining with Na + -taurocholate co-transporting polypeptide (NTCP), which is a hepatocyte basal membrane marker (36).NTCP was precisely localized in WT hepatocyte basal membranes, whereas NTCP displayed diffuse cytosolic localization in the dKO hepatocytes and this abnormality was corrected by the tKO hepatocytes, at least partially (Fig. 4A).We also stained for bile salt export pump, which is located on the bile canaliculi membrane (apical membrane) (37).In normal liver section, bile canalicular should be stained as dots (cross section) and networks (longitudinal section).We can see the number of networks is more than that of dots in WT and the tKO mouse liver sections (Fig. 4B).However, the networks are greatly reduced in the dKO mouse liver sections (Fig. 4B), suggesting an impairment.Also, we measured total bile acids and found that the WT and tKO mice had significantly lower bile acids than that of the dKO mice (Fig. S6).Collectively, SMSr deficiency reversed the liver impairment, at least partially.We stained 6-month-old female mouse liver sections with trichrome and found that the dKO but not WT or the tKO mouse livers had fibrosis (Figs.4C and S7).We quantified collagen fibers stained with trichrome and found that the dKO mouse livers had significantly higher levels of collagen fibers than that of WT mice and this effect was greatly reversed in the tKO mouse livers (Fig. 4E), indicating that SMSr deficiency could reverse liver fibrosis caused by GluCer accumulation (34).We observed that the dKO mice suffered from liver cirrhosis, and it was technically challenging to obtain primary hepatocytes using the collagenase perfusion procedure when the dKO mice were 4 months or older.By contrast, we had no difficulties obtaining primary hepatocytes from the tKO mouse livers, regardless of age.One obvious pathology of 1-year-old dKO mice was that they had liver tumors of various sizes, whereas 1year-old WT or the tKO mice did not have any tumors (Figs.4D  and S8).Similar phenotypes were observed in male mice (data not shown).We also measured ALT and AST in plasma and found that the tKO mice had significantly lower ALT and AST activity levels than that of the dKO mice (Fig. 4, F and G).

Effect of SMSr deficiency on the expression of genes related to inflammation, fibrosis, and tumorigenesis
We reported previously that the dKO mouse liver expressed many inflammation-, fibrosis-, and tumorigenesis-related genes, such as upregulation of transforming growth factor β1 (Tgfβ1), Tnfα, Il6, Il1, Colα1, Pdfgrs, and Mycn compared with WT mice and we attributed this to SMS1 deficiency-mediated GluCer accumulation (34).In current study, we isolated primary hepatocytes from chow-fed dKO and tKO female mice and performed total RNA-seq.We found that, in comparison with the dKO hepatocytes, the tKO hepatocytes have 81 upregulated genes and 141 downregulated genes (Fig. 5A).Among the 141 downregulated genes, we listed 53 which are related with NASH, fibrosis, the tumorigenesis, together with that of WT mice (Fig. 5B).We performed real-time PCR for Tgfβ1, Tnfα, Colα1, Pdfgrs, Timp-1, CCND2, and Mycn, the key factors for NASH, liver fibrosis, and liver tumor (38) in WT, the dKO, and the tKO mouse livers.All showed similar trends of increased expression in livers from the dKO mice that were reversed, at least partially, to WT levels in the tKO mice (Fig. 5C).We performed immunoblotting for TGFβ1 (a 43 kDa precursor and a 12.5 kDa active form) and collagen 1α1 in the liver.The levels of all proteins were significantly decreased in the tKO mouse livers than those in the dKO mouse livers (Fig. 5D).Inflammatory cytokine array analysis indicated that approximately 20 cytokines were downregulated in the tKO plasma than that in the dKO plasma (Fig. 5E).These combined results indicate that SMSr deficiency attenuates NASH and fibrosis caused by a deficiency of both SMS1 and SMS2.

Effect of SMSr, as a PE-PLC, deficiency on NAFLD PE attenuates TGFβ1-mediated fibrosis and tumorigenesis
To observe the direct effect of PE (mimicking SMSr deficiency), we treated the dKO mice with PE (i.p.) (10 mg/kg) every day for 7 days to mimic triple deficiency.Immunoblotting analysis showed that the active form of TGFβ1 (12.5 kDa) was dramatically reduced in PE-treated dKO mouse plasma (Fig. 6A).In PE-treated dKO liver, there was no change in the level of TGFβ1 precursor (43 kDa), but collagen 1α1 was significantly reduced (Fig. 6A), suggesting that PE supplement-mediated reduction of TGFβ1 in the plasma could mitigate liver fibrosis.
We next examined the effects of PE supplementation on LX2 cells, a hepatic stellate cell (HSC) line, by treating the cells with TGFβ1 (10 ng/ml) with or without 20 μM PE.The results showed that PE supplementation in LX-2 cells significantly reduced TGFβ1-induced production of fibrosis proteins (i.e., collagen 1α1, platelet-derived growth factor receptor (PDGFR)α, and PDGFRβ) (Fig. 6B), suggesting that PE supplementation could rescue liver fibrosis caused by TGFβ1 stimulation.
We found that the dKO but not WT and the tKO mice develop liver tumors (Figs.4D and S6).This could be related to the TGFβ1-Wnt/β-catenin pathway (39,40).TGFβ1 upregulation in Sms1/Sms2-dKO liver could result in β-catenin translocation from hepatocyte plasma membrane into the nucleus, thereby initiating tumorigenesis (34).We detected significant accumulation of β-catenin in the plasma membrane of the tKO mouse liver and a significant reduction of β-catenin in the corresponding cytosol and nucleus (Fig. 6C) compared with the levels in the Sms1/Sms2-dKO mouse liver.We also detected significant accumulation of cadherin in the plasma membrane of the tKO liver (Fig. 6C).This result indicates that SMSr deficiency could reverse TGFβ1-Wnt/β-catenin-mediated liver tumorigenesis.We injected the dKO mice with PE (i.p.) to confirm these results and found that PE treatment increased β-catenin and cadherin on liver plasma membrane and decreased β-catenin in the cytosol and nucleus compared with the levels in the control (Fig. 6D).This result indicated that PE can reduce cytosolic and nuclear levels of β-catenin.

Association of liver SMSr/PE-PLC activity and plasma PE with human NASH
We evaluated the relationship between PE-PLC/SMSr and NASH in humans.Liver samples from NASH patients and controls were immunostained with SMSr antibody.SMSr protein expression was significantly higher in the diseased livers (Fig. 7, A and B).NASH patients displayed a trend of reduced PE in the plasma, but it was not statistically significant (Fig. 7C).We also measured PC levels and found that the difference was not significant (Fig. S9A).However, the plasma PE/PC ratio was significantly lower in NASH patients than in controls (Fig. 7D).We also found that NASH patients had significantly higher levels of plasma TNFα and TGFβ1 (Fig. 7, E and F).Importantly, plasma TNFα levels were negatively correlated with PE (Fig. 7G), as were TGFβ1 and PE (Fig. 7H).The correlation of PC with TNFα or TGFβ was not significant (Fig. S9, B and C).The combined results indicate that plasma PE levels are negatively associated with human NASH.

Discussion
This is the first functional study of SMSr as a PE-PLC.First, we provide evidence that in comparison with WT mice, SMSr/ PE-PLC deficiency can attenuate high fat/fructose-induced NAFLD, including fatty liver and NASH.Second, the current study is a continuation of our previous study (34) which indicated that the SMS1 deficiency-mediated GluCer accumulation in mice results in NAFLD, including fatty liver, NASH, and liver fibrosis as well as tumor.We found that the SMSr deficiency can reverse GluCer accumulation-mediated NASH, liver fibrosis, and tumor formation and provided evidence that PE-PLC/SMSr deficiency-mediated PE accumulation is the key factor mediating these beneficial effects.SMSr transfers phosphorylethanolamine from PE to ceramide in vitro to produce CPE (3).Although Smsr is expressed ubiquitously (4), we and others showed that CPE levels are extremely low in the liver and plasma (4, 5) and undetectable in most mammalian tissues (unpublished observation).Mouse Smsr gene KO (4, 5) or overexpression (6) had little effect on CPE.Therefore, mammalian SMSr is not a functional CPE synthase in vivo.The current study is an investigation of the effect of SMSr/PE-PLC-mediated PE changes on NAFLD.
PE can be regulated by SMSr/PE-PLC.Tissue PE levels are controlled by biosynthesis and catabolism.In addition to a common biosynthesis pathway (the Kennedy pathway) ( 16), mammalian tissues have PC-specific PLCs and PI-specific PLCs that hydrolyze PC and PI to regulate steady-state levels of PC and PI (14,41,42).We recently reported that SMSr can hydrolyze PE in the absence of ceramide, and SMSr expression in mouse liver can affect PE levels (6).We confirmed that SMSr/PE-PLC overexpression significantly induced liver PE-PLC activity and reduced liver PE levels (Fig. 1, A and C), whereas SMSr/PE-PLC deficiency significantly reduced liver PE-PLC activity and increased liver PE levels (Fig. 1, B and C).We observed that SMSr/PE-PLC deficiency did not completely eliminate PE-PLC activity (Fig. 1B), suggesting that other enzymes can hydrolyze PE to generate DAG.PE is essential for autophagy, cell division, and protein folding, and is a precursor for several protein modifications (17,18).PE promotes nonlamellar membrane structures and modulates membrane curvature and fluidity (43).Abnormally high or low PE/PC ratios affect energy metabolism in various tissues and are linked to disease progression (21).
Although PE is important in several aspects of metabolic diseases, the mechanisms linking PE to metabolic diseases are mostly unknown.We observed that SMSr/PE-PLC deficiencymediated PE accumulation in liver attenuated high-fat diet/ fructose-induced body weight gain, hyperlipidemia, fatty liver, and NASH.Liver RNA-seq analysis clearly indicates that SMSr deficiency is closely related with lipid metabolic process, lipid transport, and inflammation (Fig. 2, B and C).We then evaluated mRNA levels of certain key factors, including PPARγ2, FAS, Fas27, and TNFα in NAFLD and found that SMSr deficiency can significantly reduce all of them.Further study indicated that Fsp27, TNFα, IL-6, and more than 20 inflammatory cytokines were reduced (Fig. 2, D-F).Fsp27 is expressed in the livers of diet-induced obese mice (44,45).Fsp27 inhibition mitigates diet-induced NASH in mice (46).TNFα and IL-6 have been identified as a major regulator of inflammatory responses and is involved in the pathogenesis of many inflammatory diseases (47), and they participate in the development and progression of NAFLD (48).However, we did not observe fibrosis in both WT and SMSr/PE-PLC deficient mice.The stages of NASH-associated fibrosis range from absent (stage F0) to cirrhosis (stage 4), with stages F2-F4 considered to be clinically significant and stages F3-F4 considered to be advanced fibrosis (35).The reason that we did not observe fibrosis in both WT and the KO mice could be due to the feeding time not long enough.
We recently reported that blocking the liver SM synthase (SMS1 and SMS2 double deficiency) caused NASH, fibrosis, and liver tumor.All these phenotypes were related to GluCer accumulation caused by SMS1 deficiency (34).We hypothesized that SMSr/PE-PLC deficiency could reverse the metabolic phenotypes observed in SMS1/SMS2 double-deficient mice.Therefore, we prepared SMS1/SMS2/SMSr tripledeficient mice, and observed that triple deficiency increased liver total PE (Fig. 3A) and individual PE (Fig. 3B) compared with the SMS1/SMS2 double deficiency.We also found that the triple deficiency significantly reduced liver GluCer levels (Fig. 3, C and D), and PE supplementation in vivo significantly inhibited GCS activity in the liver (Fig. 3E).Many studies report that GCS inhibition mitigates hepatic steatosis in mouse models (33,49,50), and GCS is highly expressed in the liver of NASH patients (34).Simple fatty liver is considered benign, but the initiation of inflammation and subsequent fibrosis and cirrhosis are critical steps in NAFLD with NASH (51)(52)(53).SMSr could be a new therapeutic target for these debilitating diseases.
RNA-seq and protein dot blot analysis indicated that more than 20 inflammatory cytokines, including TNFα and TGFβ1, were upregulated in the dKO mouse livers compared with WT livers, and the tKO brought these cytokine levels into normal ranges (Fig. 5, B and C).TGFβ1 is an important cytokine that performs many cellular functions, including inflammation, fibrosis, and tumorigenesis (54).The TGFβ1 precursor has 391 amino acids, with a molecular weight of 43 kDa (55).The mature form of TGFβ1 is a dimer (25 KDa) with a disulfide bond that can be reduced to form a secretory molecule of TGFβ1 with a molecular weight of 12.5 kDa (55).The tKO mice had significantly lower levels of the active form of TGFβ1 in the circulation, and significantly lower collagen 1α1, which is directly associated with fibrosis, in the liver than those of dKO mice (Fig. 5D).Administration of PE to the dKO mice significantly reduced TGFβ1 and collagen 1α1 levels (Fig. 6A).PE-treated HSC significantly attenuated TGFβ1-mediated upregulation of collagen 1α1 and PDGFR α and β (Fig. 6B), which are key factors for liver fibrosis (56).Thus, SMSr deficiency or PE administration effectively prevented TGFβ1mediated overexpression of liver fibrosis genes.
Why does PE accumulation prevent the transition from fatty liver into NASH and fibrosis?PE improves the processing of glycosylphosphatidylinositol-associated proteins (57).glycosylphosphatidylinositol moieties have crucial roles in driving transient, relatively ordered membrane domains rich in sphingolipids and cholesterol (called lipid rafts) to their target regions (58)(59)(60).PE also reduces endoplasmic reticulum (61) stress, which is closely related to NASH and liver fibrosis (62).Autophagy is a critical pathway for the degradation of intracellular components by lysosomes.Autophagy can regulate hepatocellular lipid accumulation by selective degradation; thus, autophagy has been implicated in a protective role during NAFLD and NASH (63).PE increases autophagic flux because autophagy depends on PE for autophagosome formation (64).
Another key finding of the current study was that SMSr deficiency prevented tumorigenesis caused by GluCer Effect of SMSr, as a PE-PLC, deficiency on NAFLD accumulation (Figs.4D and S6).It is known that only a small portion of liver fibrosis can result in tumors (52,65) and TGFβ1 is one of the factors promoting this transformation (39,40).TGFβ1 can disrupt the cell plasma membrane cadherin/βcatenin complex and promote β-catenin nuclear translocation (66).We previously showed that liver SMS1 deficiency affects the cellular distribution of β-catenin, which is transferred from the plasma membrane into the nucleus, to stimulate tumor gene expression (34).We found that the tKO mice and PEsupplemented dKO (mimicking the tKO) mice displayed significant disruption of β-catenin transfer to the nucleus (Fig. 6, C and D), which was typically observed in the dKO mice (34).Thus, SMSr deficiency or PE administration effectively prevented the transition of liver fibrosis to the development of liver tumor.
The field of cancer therapy has different views about PE.The plasma membrane of normal cells is an asymmetric lipid bilayer with PE in the inner leaflet, whereas PE is located in the outer leaflet in cancer cells (67).PE-binding peptides and some small molecules (e.g., ophiobolin A) bind to PE and induce pore formation, subsequently leading to cell lysis (68).It has been suggested that targeting PE on endothelial cells in tumor vasculature could achieve antiangiogenesis and inhibit cancer development (67).Although we cannot fully explain our observation that SMSr deficiency-mediated PE accumulation prevented rather than promoted tumorigenesis, PE accumation related to the enhencement of β-catenin menbrane binding could have a dominant effect.This deserves further study.
To evaluate the relevance of the current study for human NASH, we performed immunostaining to examine SMSr protein mass in the livers of NASH patients and controls.We found that NASH patients had significantly higher SMSr protein mass compared with controls (Fig. 7, A and B).We also found that NASH patients had lower plasma PE and PE/ PC ratio, although the former was not statistically significant (Fig. 7, C and D).The plasma levels of two inflammatory cytokines, TNFα and TGFβ1, were significantly higher in NASH patients compared with controls (Fig. 7, E and F), and both were negatively associated with plasma PE levels (Fig. 7, G  and H).
However, the relationship between liver PE and NAFLD is controversial.On the one hand, liver PE levels were reduced in NAFLD (69).On the other hand, elevated liver PE levels increased susceptibility to the disease progression of obesity associated NAFLD (70).The controverse also exist on PE/PC ratio.Decreasing or increasing PE/PC ratio can result in NAFLD (71).Even increased plasma levels PE were associated with NAFLD (72).Thus, there must be a proper ratio of both phospholipids in the circulation; otherwise, the metabolic balance could be compromised and NAFLD/NASH could be promoted.Although we cannot provide the proper ratio from current study, plasma PE seems to be a protective factor for the development of NASH.
In summary, we found that SMSr/PE-PLC deficiency caused PE accumulation, which can attenuate NAFLD, including fatty liver, NASH, liver fibrosis, and liver tumor formation, under different conditions.This suggests that inhibition of SMSr/PE-PLC could be a new strategy for the treatment of NAFLD.

High-fat diet/fructose study
Three-month-old mice were fed with a high-fat diet (Envigo, Cat# TD88137) with or without fructose in drinking water for 16 weeks (5-6 mice each group).Mouse body weight was measured every week.Plasma triglyceride (Triglyceride kit, Thermo Fisher Scientific), plasma cholesterol (Cholesterol kit, Fujifilm), and liver triglyceride were measured.

Real-time PCR
Total RNA was isolated from the liver using TRIzol according to the manufacturer's instructions.Complementary DNA was synthesized using high-capacity complementary DNA reverse transcription kits (Applied Biosystems).Realtime PCR was performed on the StepOnePlus Real-time PCR system using the SYBR Green Master Mix System (Applied Biosystems).Primers used for real-time PCR are listed in Table S1.

Western blot analysis
Tissue or cell homogenates were subjected to Western blotting as described previously (74).The following primary antibodies were used: Fat-specific protein 27 (Fsp27, Novus Biologicals, Catalog No. NB100-430SS); Second primary production algorithm round robin (PPARr2, Abcam, Catalog No.

Hematoxylin and eosin (H&E) and trichrome staining
Mouse livers were dissected and fixed overnight in 4% formalin.The tissues were embedded in paraffin and then sliced into 5-μm thick sections.Each slice was deparaffinized and stained with H&E or trichrome staining.The liver frozen sections were stained with Oil Red O.

Immunofluorescence staining
Mouse livers were fixed in 4% formalin overnight at 4 C before preparation of 5-μm thick paraffin sections.The sections were deparaffinized in xylene, rehydrated in a gradient series of ethanol, and subjected to high-temperature antigen retrieval in 50 mM Tris-HCl (pH 9.0) and 1 mM EDTA.The sections were permeabilized and blocked in Tris-HCl with 0.5% Triton X-100 and 5% horse serum.Then, the sections were incubated overnight at 4 C with the primary antibodies for NTCP (Invitrogen Catalog No. PA5-80001), bile salt export pump (Invitrogen Catalog No. PA5-78690), or SMSr (Proteintech, Catalog No. 26815-1-AP).The slides were washed and then incubated with fluorescent-labeled secondary antibodies.

RNA sequencing (RNA-seq)
Fresh frozen liver samples that were isolated from WT and Smsr-KO mice fed with high-fat diet with fructose (in drinking water) for 16 weeks (n = 4 per group, female), were sent to Azenta Life Sciences Company for RNA extraction, library preparation, and standard RNA sequencing.Briefly, total RNA was extracted using Qiagen RNeasy Plus Universal mini kit following the manufacturer's instructions (Qiagen).RNA samples were quantified using Qubit 4 Fluorometer (Life Technologies) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies).The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit s (NEB) and sequenced on an Illumina HiSeq 4000 using a 2 × 150 bp paired-end configuration.
Hepatocyte samples were isolated from 2-month-old WT, Sms1/Sms2-dKO, and Sms1/Sms2/Smsr-tKO mice (n = 3 per group) using a two-step collagenase method.RNA was extracted from primary hepatocytes with RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.RNA integrity was checked using 2200 TapeStation system (Agilent Technologies).Then, the RNA samples were sent to BGI Genomics Company for RNA sequencing using BGISEQ-500 platform.

RNA sequencing data processing and differential expression analysis
For liver samples from WT and Smsr-KO mice, the raw data of sequence reads were trimmed using Trimmomatic (v.0.36).For isolated primary hepatocytes from WT, Sms1/Sms2-dKO, and Sms1/Sms2/Smsr-tKO mice, the raw sequencing reads were trimmed using SOAPnuke (v.1.5.2).The trimmed reads were further mapped to the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner (v.2.5.2b) and Bowtie2 (v.2.2.5), respectively.Then, the extracted gene hit counts were used for differential expression analysis using DESeq2 (v.1.30.1).For Smsr deficient and WT liver datasets, genes with an adjusted p-value <0.05 and absolute fold change >1.2 were called differentially expressed genes (DEGs) for each comparison.With DEGs, we performed Gene Ontology functional enrichment analysis using the database of GO Ontology database (http://geneontology.org/) with default parameters.The dot enrichment plot was plotted by http://www.bioinformatics.com.cn/srplot, an online platform for data analysis and visualization (false discovery rate < 0.05).For the differential expression analysis between Sms1/ Sms2-dKO and Sms1/Sms2/Smsr-tKO hepatocytes in mice, genes with an adjusted p-value <0.05 and absolute fold change >2 were identified as DEGs for comparison.

Liquid chromatography tandem mass spectrometry (LC/MS/ MS) analysis of sphingolipids
Sphingolipid levels were measured in WT and KO mouse livers by LC/MS/MS as described (75,76).

PE and PC measurement
PE and PC were measured using the Phosphatidylethanolamine Assay Kit (Fluorometric) (Biovision, Catalog No. K499) and PC Colorimetric/Fluorometric Assay Kit (Biovision, Catalog No. K576), respectively, according to the manufacturer's protocols.

PE and TGFβ1 supplementation
For in vitro analysis, LX2 cells (HSC line, Millipore Sigma, Catalog No. SCC064) were treated with 10 ng/ml TGFβ1 and then with 20 μM PE.
For in vivo analysis, 8-week-old Sms1/Sms2-dKO mice were injected with vehicle (PBS + 5% ethanol) or PE (Avanti Polar Lipids, Catalog No. 850757P) plus vehicle, 10 μg/g body weight/day, once a day for seven consecutive days.PE was dissolved in absolute ethanol to make a 20-mM stock.Before injection, we diluted the stock solution 20× times with PBS (thus the final concentration of ethanol is 5%), we then injected (i.p.) 350 μl of this solution into a mouse (25 g), then the final concentration of PE was about 10 μg/g body weight.Afterward, mouse livers were perfused with PBS and collected for real-time PCR and Western blot analyses.
Effect of SMSr, as a PE-PLC, deficiency on NAFLD Inflammatory cytokine measurement (dot blot analysis) Inflammatory cytokines were measured using the Mouse Inflammation Array C1 kit (RayBiotch).Briefly, 2-fold dilution mouse plasma samples were incubated with antibody array membranes (Dots) overnight at 4 C after blocking.Then, the membranes were incubated with a biotinylated antibody cocktail, followed with horseradish peroxidase-streptavidin for detection.The intensity of each signal was measured using ImageJ software (https://imagej.nih.gov/ij/).

Isolation of the liver plasma membrane, cytosol, and nucleus
The liver plasma membrane and nucleus were isolated using the plasma membrane protein extraction kit (BioVision, Catalog No. K268-50) and nuclear extraction reagents (Thermo Fisher Scientific, Catalog No.78833), respectively, according to the manufacturer's protocols.

Information about human samples
Human liver microarrays with normal and NASH sections were purchased from Sekisui XenoTech, LLC (Lot.2010171), and SMSr immunostaining was performed using SMSr antibody (Proteintech, Catalog No. 26815-1-AP) overnight at 4 C.Then, the sections were incubated with fluorescently labeled secondary antibodies.Human female NASH plasma and control plasma were purchased from Discovery Life Sciences.The age of NASH patients and controls are 47.7 ± 4.4 years old.

Measurement of PE-PLC activity
Mouse liver homogenate was incubated with reaction buffer (50 mM Tris-HCl pH 7.4, 140 mM NaCl, 10 mM dimethyl glutarate, and 2 mM CaCl 2 ) and nitrobenzoxadiazole labeled PE (NBD-PE, Avanti Polar Lipids, Catalog No. 810153P) in a total volume of 500 μl.The reactions were incubated in a 37 C water bath for 30 min and then stopped by adding 500 μl of chloroform:methanol (2:1 v/v) with vigorous vortexing.The lipids were extracted and dried.For analysis, the dried lipid phase was redissolved in 20 μl of chloroform and applied to a TLC plate (Silica gel, Whatman, Catalog No. P43911).NBDsubstrates and generated NBD-DAG were separated using a basic eluent (chloroform:methanol, 15:1, v/v).The TLC plate was thoroughly dried, and the NBD-lipid species were visualized under UV light.

Quantification and statistical analysis
Statistical analysis was carried out using GraphPad Prism (https://www.graphpad.com),Version 8.0.2.Each in vitro experiment was independently performed with duplicate or triplicate to ensure reproducibility.Data are shown as mean ± SD.Unpaired two-tailed Student's t test or Mann-Whitney U test were performed for two group analyses.Multiple group comparisons were tested by Kruskal-Wallis followed by Dunn post hoc multiple comparisons tests or Mann-Whitney corrected for multiple tests.Linear regression models (Fig. 7, G and H) were used to evaluate correlations between plasma PE and TNFα and between plasma PE and TGFβ1.p values of 0.05 or less were considered to be statistically significant.

Figure 1 .
Figure 1.SMSr deficiency attenuated high-fat diet/fructose-induced fatty liver in 3-month-old WT and Smsr-KO mice.Three-month-old WT and Smsr-KO female mice were fed with high-fat diet and fructose in drinking water for 16 weeks.A, measurement of liver PE-PLC activity in WT mice treated with adenovirus-SMSr and adenovirus-control.B, measurement of liver PE-PLC activity in WT and Smsr-KO mice.C, PE levels of the liver in WT mice treated with adenovirus-SMSr/adenovirus-control and WT/Smsr KO mice.WT and Smsr-KO female mice were fed with high-fat diet with fructose (in drinking water) for 16 weeks.D, body weight gain measurement, n = 7 per group.E, measurement of plasma triglyceride and cholesterol.F, measurement of liver triglyceride.G, Oil Red O staining in sectioned liver samples; the scale bar represents 50 μm.H, trichrome staining in sectioned liver samples; the scale bar represents 50 μm.Data are presented as mean ± SD, n = 5 to 7 per group.The p values of (C, E, and F) were calculated by Mann-Whitney U test.PE-PLC, phosphatidylethanolamine phospholipase C; SMSr, sphingomyelin synthase-related protein; V-null, adenovirus-control; V-SMSr, adenovirus-SMSr.

Figure 2 .
Figure 2. SMSr deficiency affects hepatic lipid metabolism and inflammation.Three-month-old WT and Smsr-KO female mice were used.A-C, bulk RNA-seq analysis of liver from WT and Smsr-KO mice fed with high-fat diet with fructose (in drinking water) for 16 weeks (n = 4 per group).A, volcano plot for differential expression analysis between WT and Smsr-KO mice.B, Gene Ontology (GO) biological process enrichment analysis of differentially expressed genes in Smsr-KO mice liver over WT mice liver.C, heatmap of differentially expressed genes related to lipid metabolic process, lipid transport, and inflammation between WT and Smsr-KO mice.D, real-time PCR analysis of the liver key regulators involved in lipid metabolism.E, Western blots for Fsp27 (WT versus Smsr-KO, p < 0.01), PPARγ-2, and fatty acid synthase (FAS).Ponceau S staining for loading control.F, inflammatory cytokine array analysis (dot blot).G, plasma TNFα and IL-6 levels measured by ELISA.Data are presented as mean ± SD, n = 3 to 7 per group.The p values of (D, F, and H) were calculated by Mann-Whitney U test.IL-6, interleukin 6; SMSr, sphingomyelin synthase-related protein; TNF, tumor necrosis factor.

Figure 3 .
Figure 3. SMSr deficiency increased liver PE and decreased liver GluCer levels under SMS1/SMS2 double deficiency.Three-month-old WT and Smsr-KO female mice were used.A and B, total and individual PE levels in the liver among WT, Sms1/Sms2-dKO, and Sms1/Sms2/Smsr-tKO mice.C and D, total and individual GluCer levels in the liver among WT, Sms1/Sms2-dKO, and Sms1/Sms2/Smsr-tKO mice.E, GluCer synthase measurement.Sms1/Sms2-double KO mice were treated with PE or vehicle for 7 days and liver GluCer synthase activity was measured and quantified.Data are presented as mean ± SD, n = 3 to 4 per group.The p values of (A-D) were calculated by Kruskal-Wallis test followed by Mann-Whitney pairwise tests (A and C) or Dunn post hoc multiple comparisons tests (B and D).The p values of (E) were calculated by Mann-Whitney U test.*p < 0.05, **p < 0.01.GluCer, glucosylceramide; PE, phosphatidylethanolamine; SMSr, sphingomyelin synthase-related protein; tKO, triple KO.

Figure 7 .
Figure 7. SMSr/PE-PLC and PE levels in NASH patients.Human liver microarrays with normal and NASH sections were purchased from Sekisui XenoTech, LLC.Human female NASH plasma and control plasma were purchased from Discovery Life Sciences.A and B, SMSr immunostaining was performed on liver sections from human NASH patients and controls.Quantification was shown by fluorogram.C and D, NASH patient and control plasma PE and PC were measured.PE and PE/PC quantification was shown by fluorogram.E and F, NASH patient and control plasma TNFα and TGFβ were measured by ELISA.G, correlation between human plasma PE and TNFα levels.H, correlation between human plasma PE and TGFβ1 levels.Data are presented as mean ± SD, n = 4 to 7 per group (B-F).The p values of (B-F) were calculated by unpaired two-tailed Student's t test.The p values of (G and H) were calculated by linear regression model.NASH, nonalcoholic steatohepatitis; PC, phosphatidylcholine; PE-PLC, phosphatidylethanolamine phospholipase C; SMSr, sphingomyelin synthase-related protein; TGFβ1, transforming growth factor β1; TNF, tumor necrosis factor.